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The C5a Receptor (C5aR) C5L2 Is a Modulator of C5aR-mediated Signal Transduction*

  • Claire E. Bamberg
    Footnotes
    Affiliations
    Pulmonary Division, Department of Pediatrics, Children's Hospital, Boston, Massachusetts 02115

    Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
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  • Charles R. Mackay
    Footnotes
    Affiliations
    Immunology and Inflammation Department, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia

    G2 Inflammation Pty. Ltd., 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia
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  • Hyun Lee
    Affiliations
    Immunology and Inflammation Department, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia
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  • David Zahra
    Affiliations
    Immunology and Inflammation Department, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia

    G2 Inflammation Pty. Ltd., 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia
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  • Jenny Jackson
    Affiliations
    Immunology and Inflammation Department, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia
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  • Yun Si Lim
    Affiliations
    Immunology and Inflammation Department, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia
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  • Peter L. Whitfeld
    Affiliations
    G2 Inflammation Pty. Ltd., 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia
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  • Stewart Craig
    Affiliations
    Pulmonary Division, Department of Pediatrics, Children's Hospital, Boston, Massachusetts 02115
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  • Erin Corsini
    Affiliations
    Pulmonary Division, Department of Pediatrics, Children's Hospital, Boston, Massachusetts 02115
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  • Bao Lu
    Affiliations
    Pulmonary Division, Department of Pediatrics, Children's Hospital, Boston, Massachusetts 02115
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  • Craig Gerard
    Correspondence
    To whom correspondence may be addressed: 320 Longwood Ave, Boston, MA 02115. Fax: 617-730-0240;
    Affiliations
    Pulmonary Division, Department of Pediatrics, Children's Hospital, Boston, Massachusetts 02115

    Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
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  • Norma P. Gerard
    Correspondence
    To whom correspondence may be addressed: 320 Longwood Ave, Boston, MA 02115. Fax: 617-730-0240;
    Affiliations
    Pulmonary Division, Department of Pediatrics, Children's Hospital, Boston, Massachusetts 02115

    Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

    Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Institutes of Health Grants HL36162 (to N. P. G.) and HL51366 (to C. G.). This work was also supported by funding from the Medical Research Council (to C. R. M.).
    1 Both authors contributed equally to this work.
Open AccessPublished:December 31, 2009DOI:https://doi.org/10.1074/jbc.M109.092106
      The complement anaphylatoxin C5a is a proinflammatory component of host defense that functions through two identified receptors, C5a receptor (C5aR) and C5L2. C5aR is a classical G protein-coupled receptor, whereas C5L2 is structurally homologous but deficient in G protein coupling. In human neutrophils, we show C5L2 is predominantly intracellular, whereas C5aR is expressed on the plasma membrane. Confocal analysis shows internalized C5aR following ligand binding is co-localized with both C5L2 and β-arrestin. Antibody blockade of C5L2 results in a dramatic increase in C5a-mediated chemotaxis and ERK1/2 phosphorylation but does not alter C5a-mediated calcium mobilization, supporting its role in modulation of the β-arrestin pathway. Association of C5L2 with β-arrestin is confirmed by cellular co-immunoprecipitation assays. C5L2 blockade also has no effect on ligand uptake or C5aR endocytosis in human polymorphonuclear leukocytes, distinguishing its role from that of a rapid recycling or scavenging receptor in this cell type. This is thus the first example of a naturally occurring seven-transmembrane segment receptor that is both obligately uncoupled from G proteins and a negative modulator of signal transduction through the β-arrestin pathway. Physiologically, these properties provide the possibility for additional fine-tuning of host defense.

      Introduction

      The complement anaphylatoxin C5a is one of the most potent inflammatory mediators of the innate immune system, with the ability to activate all classes of myeloid cells as well as cells of many other lineages. Multiple studies using experimental animals deficient in C5 or mice with targeted deletion of the C5a receptor (C5aR)
      The abbreviations used are: C5aR
      C5a receptor
      ERK
      extracellular signal-regulated kinase
      MAPK
      mitogen-activated protein kinase
      FPR
      formyl peptide receptor
      fMLP
      formylmethionylleucylphenylalanine
      mAb
      monoclonal antibody
      DAPI
      4′,6-diamidino-2-phenylindole
      PMN
      polymorphonuclear leukocyte.
      have demonstrated the critical role for this anaphylatoxin in host defense. Generation of C5a has also been associated with disease conditions, including asthma, contact sensitivity reactions, autoimmune arthritis, and sepsis (reviewed in Refs.
      • Gerard C.
      • Gerard N.P.
      ,
      • Lee H.
      • Whitfeld P.L.
      • Mackay C.R.
      ,
      • Monk P.N.
      • Scola A.M.
      • Madala P.
      • Fairlie D.P.
      ). C5a manifests its activities by interaction with two known receptors, C5aR and C5L2. Cellular stimulation of the C5aR through Gαi2, Gαi3, or Gα16 results in intracellular calcium mobilization and activation of signaling pathways including phosphatidylinositol 3-kinase, diacylglycerol, MAPK, ERK, and others (
      • Buhl A.M.
      • Avdi N.
      • Worthen G.S.
      • Johnson G.L.
      ,
      • Perianayagam M.C.
      • Balakrishnan V.S.
      • King A.J.
      • Pereira B.J.
      • Jaber B.L.
      ), (reviewed in Ref.
      • Johswich K.
      • Klos A.
      ).
      Like the C5aR, C5L2 is a putative seven-transmembrane segment protein that was identified by Ohno et al. (
      • Ohno M.
      • Hirata T.
      • Enomoto M.
      • Araki T.
      • Ishimaru H.
      • Takahashi T.A.
      ) as a cDNA with homology to the C5aR. It has similar sequence homology with the formyl peptide receptor (FPR) and the chemokine receptor chemR23. C5L2 is expressed on neutrophils, macrophages, and immature dendritic cells in coordination with the C5aR, although its mRNA is present at significantly reduced levels (
      • Ohno M.
      • Hirata T.
      • Enomoto M.
      • Araki T.
      • Ishimaru H.
      • Takahashi T.A.
      ,
      • Chen N.J.
      • Mirtsos C.
      • Suh D.
      • Lu Y.C.
      • Lin W.J.
      • McKerlie C.
      • Lee T.
      • Baribault H.
      • Tian H.
      • Yeh W.C.
      ). Expression has additionally been reported in adrenal gland, spinal cord, thyroid, liver, lung, spleen, brain, and heart (
      • Kalant D.
      • MacLaren R.
      • Cui W.
      • Samanta R.
      • Monk P.N.
      • Laporte S.A.
      • Cianflone K.
      ,
      • Gao H.
      • Neff T.A.
      • Guo R.F.
      • Speyer C.L.
      • Sarma J.V.
      • Tomlins S.
      • Man Y.
      • Riedemann N.C.
      • Hoesel L.M.
      • Younkin E.
      • Zetoune F.S.
      • Ward P.A.
      ).
      Studies of the distinct properties of C5L2-utilizing transfection systems demonstrate its ability to bind C5a with affinity similar to that of the C5aR. It binds the metabolite des-Arg C5a with higher avidity (
      • Cain S.A.
      • Monk P.N.
      ,
      • Okinaga S.
      • Slattery D.
      • Humbles A.
      • Zsengeller Z.
      • Morteau O.
      • Kinrade M.B.
      • Brodbeck R.M.
      • Krause J.E.
      • Choe H.R.
      • Gerard N.P.
      • Gerard C.
      ). In contrast to the C5aR, however, we and others (
      • Okinaga S.
      • Slattery D.
      • Humbles A.
      • Zsengeller Z.
      • Morteau O.
      • Kinrade M.B.
      • Brodbeck R.M.
      • Krause J.E.
      • Choe H.R.
      • Gerard N.P.
      • Gerard C.
      ) have demonstrated that C5L2 is devoid of the ability to couple to intracellular G proteins due to an amino acid replacement of arginine by leucine in the DRY motif located in the second intracellular domain. Following transfection in L1.2 or rat basophilic leukemia cells, which are permissive for C5a-mediated signal transduction by the C5aR, ligand binding to C5L2 failed to induce calcium mobilization and resulted in minimal receptor phosphorylation relative to the C5aR. As a result, questions have been raised whether C5L2 activates G protein-independent intracellular signaling pathways or serves an alternate, ligand scavenging or other regulatory function similar to the chemokine receptors D6, CXCR7, and DARC (
      • Graham G.J.
      • McKimmie C.S.
      ,
      • Pruenster M.
      • Rot A.
      ,
      • Sierro F.
      • Biben C.
      • Martínez-Muñoz L.
      • Mellado M.
      • Ransohoff R.M.
      • Li M.
      • Woehl B.
      • Leung H.
      • Groom J.
      • Batten M.
      • Harvey R.P.
      • Martínez-A. C.
      • Mackay C.R.
      • Mackay F.
      ). To this end, a recent study by Scola et al. (
      • Scola A.M.
      • Johswich K.O.
      • Morgan B.P.
      • Klos A.
      • Monk P.N.
      ), reported ligand-independent internalization of C5L2 in transfected rat basophilic leukemia cells, which resulted in intracellular accumulation and degradation of C5a and des-Arg C5a.
      Information gleaned from studies of mice with targeted deletion of C5L2 reveals the anti-inflammatory role of the receptor. C5L2−/− animals exhibit significantly increased inflammation in a model of pulmonary immune complex injury compared with wild type animals (
      • Scola A.M.
      • Johswich K.O.
      • Morgan B.P.
      • Klos A.
      • Monk P.N.
      ,
      • Gerard N.P.
      • Lu B.
      • Liu P.
      • Craig S.
      • Fujiwara Y.
      • Okinaga S.
      • Gerard C.
      ). In a rat model of sepsis induced by cecal ligation and puncture, antibody blockade of C5L2 was associated with a dramatic increase in circulatory interleukin-6 (
      • Gao H.
      • Neff T.A.
      • Guo R.F.
      • Speyer C.L.
      • Sarma J.V.
      • Tomlins S.
      • Man Y.
      • Riedemann N.C.
      • Hoesel L.M.
      • Younkin E.
      • Zetoune F.S.
      • Ward P.A.
      ). An independently generated line of C5L2-deficient mice yielded contrasting results, that C5L2 is not only a positive regulator of the C5aR, but that it is also critical for signaling by both C5a and C3a (
      • Chen N.J.
      • Mirtsos C.
      • Suh D.
      • Lu Y.C.
      • Lin W.J.
      • McKerlie C.
      • Lee T.
      • Baribault H.
      • Tian H.
      • Yeh W.C.
      ). Among the signaling pathways affected were MAPK, ERK, and protein kinase B/Akt. Yet another study showed that genetic deficiency- or antibody-mediated blockade of C5L2 provided modest protection from cecal ligation and puncture-mediated sepsis (
      • Rittirsch D.
      • Flierl M.A.
      • Nadeau B.A.
      • Day D.E.
      • Huber-Lang M.
      • Mackay C.R.
      • Zetoune F.S.
      • Gerard N.P.
      • Cianflone K.
      • Köhl J.
      • Gerard C.
      • Sarma J.V.
      • Ward P.A.
      ). Here, protection from “high grade” sepsis, which resulted in 100% lethality, was afforded only by the combined inhibition of C5L2 and the C5aR. This report additionally showed that signaling through C5L2 but not the C5aR, led to release of the inflammatory protein, high mobility group box 1 (HMGB1) (
      • Rittirsch D.
      • Flierl M.A.
      • Nadeau B.A.
      • Day D.E.
      • Huber-Lang M.
      • Mackay C.R.
      • Zetoune F.S.
      • Gerard N.P.
      • Cianflone K.
      • Köhl J.
      • Gerard C.
      • Sarma J.V.
      • Ward P.A.
      ). The latter data supported a proinflammatory role for C5L2.
      Because of these apparently conflicting observations, as well as complexities presented by the several mouse models described and limitations based on the availability of reagents, we turned to the human system as an independent approach to understanding the role of C5L2. Human PMNs, in particular, provide the advantage that, as both C5L2 and the C5aR are expressed endogenously, the possibility for cooperative interactions between the two receptors may be evaluated. Here, we describe our findings based on utilization of monoclonal antibodies that selectively recognize and block C5a binding to human C5L2 or the C5aR but do not cross-react. Our findings reveal C5L2 functions as an intracellular receptor, which is activated as a consequence of C5aR activation. Activation of C5L2 results in inhibition of C5aR-β-arrestin-mediated ERK1/2 activation, with no apparent alteration of G protein-mediated functions. Thus, in human PMNs, C5L2 serves as a negative modulator of C5a-C5aR-mediated ERK1/2 signal transduction.

      DISCUSSION

      The studies reported here represent the first characterization of a seven-transmembrane segment receptor that is both obligately uncoupled from intracellular G proteins and a negative modulator of β-arrestin signaling pathways in human neutrophils. Since it was initially described in 2000, elucidation of the molecular mechanism of C5L2 has been relatively enigmatic. In our initial studies, we demonstrated that C5L2 transfected into murine pre-B L1.2 cells binds both C5a and des-Arg with nm affinity, but the interaction does not induce calcium mobilization or activation of the MAPK pathway (
      • Okinaga S.
      • Slattery D.
      • Humbles A.
      • Zsengeller Z.
      • Morteau O.
      • Kinrade M.B.
      • Brodbeck R.M.
      • Krause J.E.
      • Choe H.R.
      • Gerard N.P.
      • Gerard C.
      ). In contrast, L1.2 cells transfected with the C5aR exhibit robust signaling. We further showed that the deficiency of C5L2 in coupling to heterotrimeric G proteins is due to an amino acid replacement in the highly conserved DRY sequence at the end of the third transmembrane domain (
      • Okinaga S.
      • Slattery D.
      • Humbles A.
      • Zsengeller Z.
      • Morteau O.
      • Kinrade M.B.
      • Brodbeck R.M.
      • Krause J.E.
      • Choe H.R.
      • Gerard N.P.
      • Gerard C.
      ). Similar results were reported for the behavior of C5L2 transfected in rat basophilic leukemia cells (
      • Cain S.A.
      • Monk P.N.
      ).
      Data based on in vivo studies using mice with a targeted deletion of C5L2 revealed an anti-inflammatory role for this C5a receptor. In models of immune complex alveolitis, autoimmune arthritis, and contact sensitivity reactions, deficiency of C5L2 results in exacerbation of the injury, where deficiency of the C5aR is protective (
      • Gerard N.P.
      • Lu B.
      • Liu P.
      • Craig S.
      • Fujiwara Y.
      • Okinaga S.
      • Gerard C.
      ,
      • Höpken U.E.
      • Lu B.
      • Gerard N.P.
      • Gerard C.
      ,
      • Ji H.
      • Ohmura K.
      • Mahmood U.
      • Lee D.M.
      • Hofhuis F.M.
      • Boackle S.A.
      • Takahashi K.
      • Holers V.M.
      • Walport M.
      • Gerard C.
      • Ezekowitz A.
      • Carroll M.C.
      • Brenner M.
      • Weissleder R.
      • Verbeek J.S.
      • Duchatelle V.
      • Degott C.
      • Benoist C.
      • Mathis D.
      ,
      • Tsuji R.F.
      • Kawikova I.
      • Ramabhadran R.
      • Akahira-Azuma M.
      • Taub D.
      • Hugli T.E.
      • Gerard C.
      • Askenase P.W.
      ). Isolated C5L2−/− mouse bone marrow cells exhibit greater chemotactic responses to C5a compared with cells from wild type animals, supporting a direct involvement of C5L2 on inflammatory cells (
      • Gerard N.P.
      • Lu B.
      • Liu P.
      • Craig S.
      • Fujiwara Y.
      • Okinaga S.
      • Gerard C.
      ). Based on these findings, coupled with observations of coordinate expression of the two receptors, we hypothesized that C5L2 serves a modulatory role on C5a-C5aR-mediated activities.
      Confounding our investigations, however, a report of the phenotype of an independently generated line of C5L2-deficient mice contained divergent, and in some cases, completely opposing data (
      • Chen N.J.
      • Mirtsos C.
      • Suh D.
      • Lu Y.C.
      • Lin W.J.
      • McKerlie C.
      • Lee T.
      • Baribault H.
      • Tian H.
      • Yeh W.C.
      ,
      • Okinaga S.
      • Slattery D.
      • Humbles A.
      • Zsengeller Z.
      • Morteau O.
      • Kinrade M.B.
      • Brodbeck R.M.
      • Krause J.E.
      • Choe H.R.
      • Gerard N.P.
      • Gerard C.
      ,
      • Gerard N.P.
      • Lu B.
      • Liu P.
      • Craig S.
      • Fujiwara Y.
      • Okinaga S.
      • Gerard C.
      ). Further, additional studies with transfected cells indicated the ability of C5L2 to bind and transduce signals in response to C3a, C4a, and their des-arginine derivatives (
      • Kalant D.
      • MacLaren R.
      • Cui W.
      • Samanta R.
      • Monk P.N.
      • Laporte S.A.
      • Cianflone K.
      ,
      • Cain S.A.
      • Monk P.N.
      ,
      • Cui W.
      • Paglialunga S.
      • Kalant D.
      • Lu H.
      • Roy C.
      • Laplante M.
      • Deshaies Y.
      • Cianflone K.
      ,
      • Kalant D.
      • Cain S.A.
      • Maslowska M.
      • Sniderman A.D.
      • Cianflone K.
      • Monk P.N.
      ). Some of these discrepancies have subsequently been resolved (
      • Johswich K.
      • Klos A.
      ,
      • Johswich K.
      • Martin M.
      • Thalmann J.
      • Rheinheimer C.
      • Monk P.N.
      • Klos A.
      ).
      As a result of these conflicting reports, and in efforts to provide a more in-depth analysis of the molecular mechanism of C5L2, we thus sought an independent approach using human cells to identify the molecular mechanisms for the actions of C5L2. We further sought to avoid the use of transfection systems because of the potential for mismatches in components of the signaling pathways to yield misleading results.
      Prior to the present report, most of the cell biological studies of C5L2 were conducted using transfected cells, and plasma membrane expression was not limiting. In neutrophils from both humans and mice, however, ligand binding and flow cytometric analyses consistently reveal negligible C5L2 surface expression (Fig. 2, A and B).
      C. E. Bamberg, S. Craig, and N. P. Gerard, unpublished observations.
      In contrast, the C5aR is abundantly expressed on the cell surface. When PMNs are permeabilized prior to immunostaining, abundant C5L2 is apparent, demonstrating the majority of this receptor is expressed in intracellular compartment(s) (Figs. 2B and 3A).
      Similar intracellular expression has been reported for several other seven-transmembrane segment receptors, including the FPR and the non-G protein-coupled chemokine receptors D6 and CXCR7 (
      • Graham G.J.
      • McKimmie C.S.
      ,
      • Videm V.
      • Strand E.
      ,
      • Hartmann T.N.
      • Grabovsky V.
      • Pasvolsky R.
      • Shulman Z.
      • Buss E.C.
      • Spiegel A.
      • Nagler A.
      • Lapidot T.
      • Thelen M.
      • Alon R.
      ). In the case of the FPR, cellular activation by phorbol ester is a significant mechanism for inducing surface expression (
      • Videm V.
      • Strand E.
      ); however, it does not change the distribution of C5L2. The chemokine-scavenging receptor D6 recycles to the plasma membrane where it binds ligands and promotes their clearance in the absence of signaling (
      • Weber M.
      • Blair E.
      • Simpson C.V.
      • O'Hara M.
      • Blackburn P.E.
      • Rot A.
      • Graham G.J.
      • Nibbs R.J.
      ). Scola et al. (
      • Scola A.M.
      • Johswich K.O.
      • Morgan B.P.
      • Klos A.
      • Monk P.N.
      ), recently reported a ligand-scavenging activity for C5L2 in transfected rat basophilic leukemia cells; however, these cells also exhibit plasma membrane expression of the receptor and little or no endogenous C5aR. In neutrophils, our data indicate a similar mechanism does not appear significant since inhibition of C5L2 by blocking mAbs does not alter either the uptake or internalization of C5a (Fig. 4, A and B). While such a scavenging mechanism is formally possible in endogenously expressing cells under circumstances in which C5L2 is exposed to the extracellular milieu, the present work indicates it is at most minimal in human PMNs.
      CXCR7 has been shown to lie just below the plasma membrane in human T lymphocytes and serves to modify CXCR4-mediated expression of adhesion molecules (
      • Hartmann T.N.
      • Grabovsky V.
      • Pasvolsky R.
      • Shulman Z.
      • Buss E.C.
      • Spiegel A.
      • Nagler A.
      • Lapidot T.
      • Thelen M.
      • Alon R.
      ). In resting human PMNs, C5L2 appears in granular structures throughout the cytoplasm (Fig. 3). Like CXCR7, blocking antibodies against C5L2 are able to access the receptor, despite negligible surface expression. When neutrophils are activated with C5a, both C5L2 and C5aR appear to be translocated to the same compartments. One consequence of C5L2 activation is a dramatic suppression of C5a-mediated chemotaxis, with no concomitant change in mobilization of intracellular calcium (Fig. 5, A and B). Analysis of the signal transduction pathway reveals enhanced C5a-mediated ERK1/2 activation in the presence of C5L2 blockade compared with the response elicited in cells in which both receptors are functional (Fig. 5, C and D). Similarly, bone marrow cells from C5L2-deficient mice exhibit significantly greater ERK1/2 activation following C5a stimulation than cells from wild type animals.5 Although the mechanism of CXCR7-mediated activation is not yet entirely clear, our studies indicate that CXCL12 activates this receptor exclusively through the β-arrestin pathway (
      • Rajagopal S.
      • Kim J.
      • Ahn S.
      • Craig S.
      • Lam C.M.
      • Gerard N.P.
      • Gerard C.
      • Lefkowitz R.J.
      ).
      In resting neutrophils, confocal analysis reveals C5L2 distributed throughout the cytoplasm co-localized with β-arrestin, particularly in regions juxtaposed to the nucleus (Fig. 6A). Co-immunoprecipitation of C5L2 with β-arrestin is consistent with molecular association of the two proteins (Fig. 6B). In resting cells, the presence of C5aR is evident at the plasma membrane. Following addition of C5a, association of both C5L2 and C5aR with β-arrestin is greatly increased. Further, the increased co-localization of C5L2 with β-arrestin that occurs following addition of C5a is blocked in cells treated with anti-C5aR antibodies (Fig. 7), a finding additionally supportive of the function of C5L2 as an intracellular receptor.
      While this manuscript was under review, two literature reports appeared demonstrating C5a-dependent redistribution of β-arrestin-GFP in cells co-transfected with C5L2 (
      • Cui W.
      • Simaan M.
      • Laporte S.
      • Lodge R.
      • Cianflone K.
      ,
      • van Lith L.H.
      • Oosterom J.
      • van Elsas A.
      • Zaman G.J.
      ). Although the functional significance to C5aR-mediated responses was not shown, these studies confirm our findings in human PMNs. One of the reports additionally corroborated their result with a β-galactosidase fragment complementation assay, in which the receptor was labeled with a mutated peptide of β-galactosidase and β-arrestin was labeled with a corresponding deletion mutant of the enzyme (
      • van Lith L.H.
      • Oosterom J.
      • van Elsas A.
      • Zaman G.J.
      ). Following cellular activation generation of a chemiluminescent signal indicated receptor-mediated association with β-arrestin. Importantly, while these investigators demonstrated recruitment of β-arrestin to C5L2 following C5a stimulation, they also showed an absence of ERK1/2 activation in these cells, a result also consistent with our findings in PMNs because we find ERK1/2 activation results from activation of the C5aR.
      Taken together, our data support a model depicted schematically in Fig. 8 in which C5L2 functions as an intracellular receptor, becoming activated only after ligand binding to the C5aR. Activation of the receptors induces their association with β-arrestin, which, in the case of the C5aR, results in activation of ERK1/2. The C5L2-β-arrestin complex inhibits ERK1/2, and the net signal is a result of the balance of the two. By analogy, β2-adrenergic and AT1a receptors bearing mutated DRY sequences have been shown to induce distinct conformations for β-arrestin compared with their wild type counterparts (
      • Shukla A.K.
      • Violin J.D.
      • Whalen E.J.
      • Gesty-Palmer D.
      • Shenoy S.K.
      • Lefkowitz R.J.
      ). These mutated receptors are completely uncoupled from G proteins but maintain their ability to activate ERK1/2. Our previous studies demonstrate the mutation in the DRY sequence determines the lack of coupling to G proteins. The ERK1/2 inhibitory property of the C5L2-β-arrestin complex represents a novel regulatory feature of C5a-mediated neutrophil activation. Understanding the molecular mechanism of this suppression provides another clue to the complex regulation of innate immunity.
      Figure thumbnail gr8
      FIGURE 8Schematic representation of the mechanism by which C5L2 negatively modulates C5a-C5aR-mediated ERK1/2 activation in human neutrophils. C5L2 functions as an intracellular receptor, becoming activated only after ligand binding to the C5aR. Receptor activation induces phosphorylation by G protein receptor kinases (GRK), facilitating their association with β-arrestin. The C5aR-β-arrestin complex activates ERK1/2, whereas the C5L2-β-arrestin complex inhibits ERK1/2, and the net signal is a result of the balance of the two.

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